Differentiation of human embryonic stem cells

ABSTRACT

The present invention provides methods to promote the differentiation of pluripotent stem cells into cells expressing markers characteristic of the pancreatic endocrine lineage that co-express PDX1, NKX6.1, but do not express CDX2 and NGN3.

CROSS REFERENCE TO RELATED APPLICATION

The present invention claims priority to application Ser. No.61/226,929, filed Jul. 20, 2009.

FIELD OF THE INVENTION

The present invention provides methods to promote the differentiation ofpluripotent stem cells into cells expressing markers characteristic ofthe pancreatic endocrine lineage that co-express PDX1, NKX6.1, but donot express CDX2 and NGN3.

BACKGROUND

Advances in cell-replacement therapy for Type I diabetes mellitus and ashortage of transplantable islets of Langerhans have focused interest ondeveloping sources of insulin-producing cells, or β cells, appropriatefor engraftment. One approach is the generation of functional β cellsfrom pluripotent stem cells, such as, for example, embryonic stem cells.

In vertebrate embryonic development, a pluripotent cell gives rise to agroup of cells comprising three germ layers (ectoderm, mesoderm, andendoderm) in a process known as gastrulation. Tissues such as, forexample, thyroid, thymus, pancreas, gut, and liver, will develop fromthe endoderm, via an intermediate stage. The intermediate stage in thisprocess is the formation of definitive endoderm. Definitive endodermcells express a number of markers, such as, HNF3 beta, GATA4, MIXL1,CXCR4 and SOX17.

Formation of the pancreas arises from the differentiation of definitiveendoderm into pancreatic endoderm. Cells of the pancreatic endodermexpress the pancreatic-duodenal homeobox gene, PDX1. In the absence ofPDX1, the pancreas fails to develop beyond the formation of ventral anddorsal buds. Thus, PDX1 expression marks a critical step in pancreaticorganogenesis. The mature pancreas contains, among other cell types,exocrine tissue and endocrine tissue. Exocrine and endocrine tissuesarise from the differentiation of pancreatic endoderm.

Cells bearing the features of islet cells have reportedly been derivedfrom embryonic cells of the mouse. For example, Lumelsky et al. (Science292:1389, 2001) report differentiation of mouse embryonic stem cells toinsulin-secreting structures similar to pancreatic islets. Soria et al.(Diabetes 49:157, 2000) report that insulin-secreting cells derived frommouse embryonic stem cells normalize glycemia in streptozotocin-induceddiabetic mice.

In one example, Hori et al. (PNAS 99: 16105, 2002) disclose thattreatment of mouse embryonic stem cells with inhibitors ofphosphoinositide 3-kinase (LY294002) produced cells that resembled βcells.

In another example, Blyszczuk et al. (PNAS 100:998, 2003) reports thegeneration of insulin-producing cells from mouse embryonic stem cellsconstitutively expressing Pax4.

Micallef et al. reports that retinoic acid can regulate the commitmentof embryonic stem cells to form PDX1 positive pancreatic endoderm.Retinoic acid is most effective at inducing Pdx1 expression when addedto cultures at day 4 of embryonic stem cell differentiation during aperiod corresponding to the end of gastrulation in the embryo (Diabetes54:301, 2005).

Miyazaki et al. reports a mouse embryonic stem cell line over-expressingPdx1. Their results show that exogenous Pdx1 expression clearly enhancedthe expression of insulin, somatostatin, glucokinase, neurogenin3, p48,Pax6, and HNF6 genes in the resulting differentiated cells (Diabetes 53:1030, 2004).

Skoudy et al. reports that activin A (a member of the TGF-β superfamily)upregulates the expression of exocrine pancreatic genes (p48 andamylase) and endocrine genes (Pdx1, insulin, and glucagon) in mouseembryonic stem cells. The maximal effect was observed using 1 nM activinA. They also observed that the expression level of insulin and Pdx1 mRNAwas not affected by retinoic acid; however, 3 nM FGF7 treatment resultedin an increased level of the transcript for Pdx1 (Biochem. J. 379: 749,2004).

Shiraki et al. studied the effects of growth factors that specificallyenhance differentiation of embryonic stem cells into PDX1 positivecells. They observed that TGF-β2 reproducibly yielded a higherproportion of PDX1 positive cells (Genes Cells. 2005 June; 10(6):503-16.).

Gordon et al. demonstrated the induction of brachyury [positive]/HNF3beta [positive] endoderm cells from mouse embryonic stem cells in theabsence of serum and in the presence of activin along with an inhibitorof Wnt signaling (US 2006/0003446A1).

Gordon et al. (PNAS, Vol 103, page 16806, 2006) states “Wnt andTGF-beta/nodal/activin signaling simultaneously were required for thegeneration of the anterior primitive streak”.

However, the mouse model of embryonic stem cell development may notexactly mimic the developmental program in higher mammals, such as, forexample, humans.

Thomson et al. isolated embryonic stem cells from human blastocysts(Science 282:114, 1998). Concurrently, Gearhart and coworkers derivedhuman embryonic germ (hEG) cell lines from fetal gonadal tissue(Shamblott et al., Proc. Natl. Acad. Sci. USA 95:13726, 1998). Unlikemouse embryonic stem cells, which can be prevented from differentiatingsimply by culturing with Leukemia Inhibitory Factor (LIF), humanembryonic stem cells must be maintained under very special conditions(U.S. Pat. No. 6,200,806; WO 99/20741; WO 01/51616).

D'Amour et al. describes the production of enriched cultures of humanembryonic stem cell-derived definitive endoderm in the presence of ahigh concentration of activin and low serum (Nature Biotechnology 2005).Transplanting these cells under the kidney capsule of mice resulted indifferentiation into more mature cells with characteristics of someendodermal organs. Human embryonic stem cell-derived definitive endodermcells can be further differentiated into PDX1 positive cells afteraddition of FGF-10 (U.S. 2005/0266554A1).

D'Amour et al. (Nature Biotechnology—24, 1392-1401 (2006)) states: “Wehave developed a differentiation process that converts human embryonicstem (hES) cells to endocrine cells capable of synthesizing thepancreatic hormones insulin, glucagon, somatostatin, pancreaticpolypeptide and ghrelin. This process mimics in vivo pancreaticorganogenesis by directing cells through stages resembling definitiveendoderm, gut-tube endoderm, pancreatic endoderm and endocrine precursoren route to cells that express endocrine hormones”.

In another example, Fisk et al. reports a system for producingpancreatic islet cells from human embryonic stem cells (US2006/0040387A1). In this case, the differentiation pathway was dividedinto three stages. Human embryonic stem cells were first differentiatedto endoderm using a combination of sodium butyrate and activin A. Thecells were then cultured with TGF-β antagonists such as Noggin incombination with EGF or betacellulin to generate PDX1 positive cells.The terminal differentiation was induced by nicotinamide.

In one example, Benvenistry et al. states: “We conclude thatover-expression of PDX1 enhanced expression of pancreatic enrichedgenes, induction of insulin expression may require additional signalsthat are only present in vivo” (Benvenistry et al, Stem Cells 2006;24:1923-1930).

In another example, Grapin-Botton et al. states: “Early activation ofNgn3 almost exclusively induced glucagon+ cells while depleting the poolof pancreas progenitors. As from E11.5, PDX1 progenitors becamecompetent to differentiate into insulin [positive] and PP [positive]cells” (Johansson K A et al, Developmental Cell 12, 457-465, March2007).

Therefore, there still remains a significant need to develop conditionsfor establishing pluripotent stem cell lines that can be expanded toaddress the current clinical needs, while retaining the potential todifferentiate into pancreatic endocrine cells, pancreatic hormoneexpressing cells, or pancreatic hormone secreting cells. We have takenan alternative approach to improve the efficiency of differentiatinghuman embryonic stem cells toward pancreatic endocrine cells, bygenerating a population of cells expressing markers characteristic ofthe pancreatic endoderm lineage that co-express PDX1, NKX6.1, but do notexpress CDX2 and NGN3.

SUMMARY

In one embodiment, the present invention provides a method todifferentiate a population of pluripotent stem cells into a populationof cells expressing markers characteristic of the pancreatic endodermlineage that co-express PDX1, NKX6.1, but do not express CDX2 and NGN3,comprising the steps of:

-   a. Culturing the pluripotent stem cells,-   b. Differentiating the pluripotent stem cells into cells expressing    markers characteristic of the definitive endoderm lineage, and-   c. Differentiating the cells expressing markers characteristic of    the definitive endoderm lineage into cells expressing markers    characteristic of the pancreatic endoderm lineage that co-express    PDX1, NKX6.1, but do not express CDX2 and NGN3 by treating cells    expressing markers characteristic of the definitive endoderm lineage    with a first medium supplemented with FGF7, followed by culturing    the cells in a second medium supplemented with FGF7, a factor    capable of inhibiting BMP, activin A, retinoic acid, and a hedgehog    signaling pathway inhibitor.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the effect of activin A on the expression of NKX6.1, PDX1,PTF1 alpha, and ARX at stage 3 day 4, in cells treated according to themethods described in Example 1. Duplicate samples were collected forreal-time PCR analysis. The plots represent fold induction for each generelative to the control group (light grey bars). The dark gray barsrepresent cells treated with FGF7, cyclopamine-KAAD, retinoic acid, 20ng/ml activin A and noggin. The black bars represent cells treated withFGF7, cyclopamine-KAAD, retinoic acid, 50 ng/ml activin A and noggin.

FIG. 2 shows immunofluorescence images, showing the expression of NKX6.1(panels a, c, and e) and NGN3 (panels b, d and f) in cells treated withFGF7+Noggin+retinoic acid+KAAD-cyclopamine (panels a and b), or cellstreated with FGF7+Noggin+retinoic acid+KAAD-cyclopamine+20 ng/ml activinA (panels c and d), and cells treated with FGF7+Noggin+retinoicacid+KAAD-cyclopamine+Alk5 inhibitor II (panels e and f).

FIG. 3 shows immunofluorescence images, showing the expression of PDX1(panels a and c), CDX2 (panel b and d) in cells treated with DMEM-highglucose with 1% B27+FGF7+Noggin+retinoic acid+KAAD-cyclopamine+20 ng/mlactivin A (panels a and b), and cells treated with DMEM/F12-high glucosewith 1% B27+FGF7+Noggin+retinoic acid+KAAD-cyclopamine+20 ng/ml activinA (panels c and d).

DETAILED DESCRIPTION

For clarity of disclosure, and not by way of limitation, the detaileddescription of the invention is divided into the following subsectionsthat describe or illustrate certain features, embodiments orapplications of the present invention.

Definitions

Stem cells are undifferentiated cells defined by their ability at thesingle cell level to both self-renew and differentiate to produceprogeny cells, including self-renewing progenitors, non-renewingprogenitors, and terminally differentiated cells. Stem cells are alsocharacterized by their ability to differentiate in vitro into functionalcells of various cell lineages from multiple germ layers (endoderm,mesoderm and ectoderm), as well as to give rise to tissues of multiplegerm layers following transplantation and to contribute substantially tomost, if not all, tissues following injection into blastocysts.

Stem cells are classified by their developmental potential as: (1)totipotent, meaning able to give rise to all embryonic andextraembryonic cell types; (2) pluripotent, meaning able to give rise toall embryonic cell types; (3) multipotent, meaning able to give rise toa subset of cell lineages but all within a particular tissue, organ, orphysiological system (for example, hematopoietic stem cells (HSC) canproduce progeny that include HSC (self-renewal), blood cell restrictedoligopotent progenitors, and all cell types and elements (e.g.,platelets) that are normal components of the blood); (4) oligopotent,meaning able to give rise to a more restricted subset of cell lineagesthan multipotent stem cells; and (5) unipotent, meaning able to giverise to a single cell lineage (e.g., spermatogenic stem cells).

Differentiation is the process by which an unspecialized (“uncommitted”)or less specialized cell acquires the features of a specialized cellsuch as, for example, a nerve cell or a muscle cell. A differentiated ordifferentiation-induced cell is one that has taken on a more specialized(“committed”) position within the lineage of a cell. The term“committed”, when applied to the process of differentiation, refers to acell that has proceeded in the differentiation pathway to a point where,under normal circumstances, it will continue to differentiate into aspecific cell type or subset of cell types, and cannot, under normalcircumstances, differentiate into a different cell type or revert to aless differentiated cell type. De-differentiation refers to the processby which a cell reverts to a less specialized (or committed) positionwithin the lineage of a cell. As used herein, the lineage of a celldefines the heredity of the cell, i.e., which cells it came from andwhat cells it can give rise to. The lineage of a cell places the cellwithin a hereditary scheme of development and differentiation. Alineage-specific marker refers to a characteristic specificallyassociated with the phenotype of cells of a lineage of interest and canbe used to assess the differentiation of an uncommitted cell to thelineage of interest.

“Cells expressing markers characteristic of the definitive endodermlineage”, or “Stage 1 cells”, or “Stage 1”, as used herein, refers tocells expressing at least one of the following markers: SOX-17, GATA4,HNF3 beta, GSC, CER1, Nodal, FGF8, Brachyury, Mix-like homeobox protein,FGF4 CD48, eomesodermin (EOMES), DKK4, FGF17, GATA6, CXCR4, C-Kit, CD99,or OTX2. Cells expressing markers characteristic of the definitiveendoderm lineage include primitive streak precursor cells, primitivestreak cells, mesendoderm cells and definitive endoderm cells.

“Cells expressing markers characteristic of the pancreatic endodermlineage”, as used herein, refers to cells expressing at least one of thefollowing markers: PDX1, HNF1 beta, PTF1 alpha, HNF6, NKX6.1, or HB9.Cells expressing markers characteristic of the pancreatic endodermlineage include pancreatic endoderm cells, primitive gut tube cells, andposterior foregut cells.

“Definitive endoderm”, as used herein, refers to cells which bear thecharacteristics of cells arising from the epiblast during gastrulationand which form the gastrointestinal tract and its derivatives.Definitive endoderm cells express the following markers: HNF3 beta,GATA4, SOX17, Cerberus, OTX2, goosecoid, C-Kit, CD99, and MIXL1.

“Markers”, as used herein, are nucleic acid or polypeptide moleculesthat are differentially expressed in a cell of interest. In thiscontext, differential expression means an increased level for a positivemarker and a decreased level for a negative marker. The detectable levelof the marker nucleic acid or polypeptide is sufficiently higher orlower in the cells of interest compared to other cells, such that thecell of interest can be identified and distinguished from other cellsusing any of a variety of methods known in the art.

“Pancreatic endocrine cell”, or “pancreatic hormone expressing cell”, asused herein, refers to a cell capable of expressing at least one of thefollowing hormones: insulin, glucagon, somatostatin, and pancreaticpolypeptide.

Isolation, Expansion and Culture of Pluripotent Stem CellsCharacterization of Pluripotent Stem Cells

Pluripotent stem cells may express one or more of the stage-specificembryonic antigens (SSEA) 3 and 4, and markers detectable usingantibodies designated Tra-1-60 and Tra-1-81 (Thomson et al., Science282:1145, 1998). Differentiation of pluripotent stem cells in vitroresults in the loss of SSEA-4, Tra 1-60, and Tra 1-81 expression (ifpresent) and increased expression of SSEA-1. Undifferentiatedpluripotent stem cells typically have alkaline phosphatase activity,which can be detected by fixing the cells with 4% paraformaldehyde, andthen developing with Vector Red as a substrate, as described by themanufacturer (Vector Laboratories, Burlingame Calif.) Undifferentiatedpluripotent stem cells also typically express Oct-4 and TERT, asdetected by RT-PCR.

Another desirable phenotype of propagated pluripotent stem cells is apotential to differentiate into cells of all three germinal layers:endoderm, mesoderm, and ectoderm tissues. Pluripotency of pluripotentstem cells can be confirmed, for example, by injecting cells into severecombined immunodeficient (SCID) mice, fixing the teratomas that formusing 4% paraformaldehyde, and then examining them histologically forevidence of cell types from the three germ layers. Alternatively,pluripotency may be determined by the creation of embryoid bodies andassessing the embryoid bodies for the presence of markers associatedwith the three germinal layers.

Propagated pluripotent stem cell lines may be karyotyped using astandard G-banding technique and compared to published karyotypes of thecorresponding primate species. It is desirable to obtain cells that havea “normal karyotype,” which means that the cells are euploid, whereinall human chromosomes are present and not noticeably altered.

Sources of Pluripotent Stem Cells

The types of pluripotent stem cells that may be used include establishedlines of pluripotent cells derived from tissue formed after gestation,including pre-embryonic tissue (such as, for example, a blastocyst),embryonic tissue, or fetal tissue taken any time during gestation,typically but not necessarily before approximately 10-12 weeksgestation. Non-limiting examples are established lines of humanembryonic stem cells or human embryonic germ cells, such as, for examplethe human embryonic stem cell lines H1, H7, and H9 (WiCell). Alsocontemplated is use of the compositions of this disclosure during theinitial establishment or stabilization of such cells, in which case thesource cells would be primary pluripotent cells taken directly from thesource tissues. Also suitable are cells taken from a pluripotent stemcell population already cultured in the absence of feeder cells. Alsosuitable are mutant human embryonic stem cell lines, such as, forexample, BG01v (BresaGen, Athens, Ga.).

In one embodiment, human embryonic stem cells are prepared as describedby Thomson et al. (U.S. Pat. No. 5,843,780; Science 282:1145, 1998;Curr. Top. Dev. Biol. 38:133 ff., 1998; Proc. Natl. Acad. Sci. U.S.A.92:7844, 1995).

Culture of Pluripotent Stem Cells

In one embodiment, pluripotent stem cells are typically cultured on alayer of feeder cells that support the pluripotent stem cells in variousways. Alternatively, pluripotent stem cells are cultured in a culturesystem that is essentially free of feeder cells, but nonethelesssupports proliferation of pluripotent stem cells without undergoingsubstantial differentiation. The growth of pluripotent stem cells infeeder-free culture without differentiation is supported using a mediumconditioned by culturing previously with another cell type.Alternatively, the growth of pluripotent stem cells in feeder-freeculture without differentiation is supported using a chemically definedmedium.

For example, Reubinoff et al (Nature Biotechnology 18: 399-404 (2000))and Thompson et al (Science 6 Nov. 1998: Vol. 282. no. 5391, pp.1145-1147) disclose the culture of pluripotent stem cell lines fromhuman blastocysts using a mouse embryonic fibroblast feeder cell layer.

Richards et al, (Stem Cells 21: 546-556, 2003) evaluated a panel of 11different human adult, fetal and neonatal feeder cell layers for theirability to support human pluripotent stem cell culture. Richards et al,states: “human embryonic stem cell lines cultured on adult skinfibroblast feeders retain human embryonic stem cell morphology andremain pluripotent”.

US20020072117 discloses cell lines that produce media that support thegrowth of primate pluripotent stem cells in feeder-free culture. Thecell lines employed are mesenchymal and fibroblast-like cell linesobtained from embryonic tissue or differentiated from embryonic stemcells. US20020072117 also discloses the use of the cell lines as aprimary feeder cell layer.

In another example, Wang et al (Stem Cells 23: 1221-1227, 2005)discloses methods for the long-term growth of human pluripotent stemcells on feeder cell layers derived from human embryonic stem cells.

In another example, Stojkovic et at (Stem Cells 2005 23: 306-314, 2005)disclose a feeder cell system derived from the spontaneousdifferentiation of human embryonic stem cells.

In a further example, Miyamoto et at (Stem Cells 22: 433-440, 2004)disclose a source of feeder cells obtained from human placenta.

Amit et at (Biol. Reprod 68: 2150-2156, 2003) discloses a feeder celllayer derived from human foreskin.

In another example, Inzunza et at (Stem Cells 23: 544-549, 2005)disclose a feeder cell layer from human postnatal foreskin fibroblasts.

U.S. Pat. No. 6,642,048 discloses media that support the growth ofprimate pluripotent stem (pPS) cells in feeder-free culture, and celllines useful for production of such media. U.S. Pat. No. 6,642,048states: “This invention includes mesenchymal and fibroblast-like celllines obtained from embryonic tissue or differentiated from embryonicstem cells. Methods for deriving such cell lines, processing media, andgrowing stem cells using the conditioned media are described andillustrated in this disclosure.”

In another example, WO2005014799 discloses conditioned medium for themaintenance, proliferation and differentiation of mammalian cells.WO2005014799 states: “The culture medium produced in accordance with thepresent invention is conditioned by the cell secretion activity ofmurine cells; in particular, those differentiated and immortalizedtransgenic hepatocytes, named MMH (Met Murine Hepatocyte).”

In another example, Xu et at (Stem Cells 22: 972-980, 2004) disclosesconditioned medium obtained from human embryonic stem cell derivativesthat have been genetically modified to over express human telomerasereverse transcriptase.

In another example, US20070010011 discloses a chemically defined culturemedium for the maintenance of pluripotent stem cells.

An alternative culture system employs serum-free medium supplementedwith growth factors capable of promoting the proliferation of embryonicstem cells. For example, Cheon et al (BioReprodDOI:10.1095/biolreprod.105.046870, Oct. 19, 2005) disclose afeeder-free, serum-free culture system in which embryonic stem cells aremaintained in unconditioned serum replacement (SR) medium supplementedwith different growth factors capable of triggering embryonic stem cellself-renewal.

In another example, Levenstein et at (Stem Cells 24: 568-574, 2006)disclose methods for the long-term culture of human embryonic stem cellsin the absence of fibroblasts or conditioned medium, using mediasupplemented with bFGF.

In another example, US20050148070 discloses a method of culturing humanembryonic stem cells in defined media without serum and withoutfibroblast feeder cells, the method comprising: culturing the stem cellsin a culture medium containing albumin, amino acids, vitamins, minerals,at least one transferrin or transferrin substitute, at least one insulinor insulin substitute, the culture medium essentially free of mammalianfetal serum and containing at least about 100 ng/ml of a fibroblastgrowth factor capable of activating a fibroblast growth factor signalingreceptor, wherein the growth factor is supplied from a source other thanjust a fibroblast feeder layer, the medium supported the proliferationof stem cells in an undifferentiated state without feeder cells orconditioned medium.

In another example, US20050233446 discloses a defined media useful inculturing stem cells, including undifferentiated primate primordial stemcells. In solution, the media is substantially isotonic as compared tothe stem cells being cultured. In a given culture, the particular mediumcomprises a base medium and an amount of each of bFGF, insulin, andascorbic acid necessary to support substantially undifferentiated growthof the primordial stem cells.

In another example, U.S. Pat. No. 6,800,480 states “In one embodiment, acell culture medium for growing primate-derived primordial stem cells ina substantially undifferentiated state is provided which includes a lowosmotic pressure, low endotoxin basic medium that is effective tosupport the growth of primate-derived primordial stem cells. The basicmedium is combined with a nutrient serum effective to support the growthof primate-derived primordial stem cells and a substrate selected fromthe group consisting of feeder cells and an extracellular matrixcomponent derived from feeder cells. The medium further includesnon-essential amino acids, an anti-oxidant, and a first growth factorselected from the group consisting of nucleosides and a pyruvate salt.”

In another example, US20050244962 states: “In one aspect the inventionprovides a method of culturing primate embryonic stem cells. Onecultures the stem cells in a culture essentially free of mammalian fetalserum (preferably also essentially free of any animal serum) and in thepresence of fibroblast growth factor that is supplied from a sourceother than just a fibroblast feeder layer. In a preferred form, thefibroblast feeder layer, previously required to sustain a stem cellculture, is rendered unnecessary by the addition of sufficientfibroblast growth factor.”

In a further example, WO2005065354 discloses a defined, isotonic culturemedium that is essentially feeder-free and serum-free, comprising: a. abasal medium; b. an amount of bFGF sufficient to support growth ofsubstantially undifferentiated mammalian stem cells; c. an amount ofinsulin sufficient to support growth of substantially undifferentiatedmammalian stem cells; and d. an amount of ascorbic acid sufficient tosupport growth of substantially undifferentiated mammalian stem cells.

In another example, WO2005086845 discloses a method for maintenance ofan undifferentiated stem cell, said method comprising exposing a stemcell to a member of the transforming growth factor-beta (TGF-β) familyof proteins, a member of the fibroblast growth factor (FGF) family ofproteins, or nicotinamide (NIC) in an amount sufficient to maintain thecell in an undifferentiated state for a sufficient amount of time toachieve a desired result.

The pluripotent stem cells may be plated onto a suitable culturesubstrate. In one embodiment, the suitable culture substrate is anextracellular matrix component, such as, for example, those derived frombasement membrane or that may form part of adhesion moleculereceptor-ligand couplings. In one embodiment, a the suitable culturesubstrate is MATRIGEL® (Becton Dickenson). MATRIGEL® is a solublepreparation from Engelbreth-Holm Swarm tumor cells that gels at roomtemperature to form a reconstituted basement membrane.

Other extracellular matrix components and component mixtures aresuitable as an alternative. Depending on the cell type beingproliferated, this may include laminin, fibronectin, proteoglycan,entactin, heparan sulfate, and the like, alone or in variouscombinations.

The pluripotent stem cells may be plated onto the substrate in asuitable distribution and in the presence of a medium that promotes cellsurvival, propagation, and retention of the desirable characteristics.All these characteristics benefit from careful attention to the seedingdistribution and can readily be determined by one of skill in the art.

Suitable culture media may be made from the following components, suchas, for example, Dulbecco's modified Eagle's medium (DMEM), Gibco#11965-092; Knockout Dulbecco's modified Eagle's medium (KO DMEM), Gibco#10829-018; Ham's F12/50% DMEM basal medium; 200 mM L-glutamine, Gibco#15039-027; non-essential amino acid solution, Gibco 11140-050;β-mercaptoethanol, Sigma #M7522; human recombinant basic fibroblastgrowth factor (bFGF), Gibco #13256-029.

Formation of Cells Expressing Markers Characteristic of the PancreaticEndoderm Lineage from Pluripotent Stem Cells

In one embodiment, the present invention provides a method for producingcells expressing markers characteristic of the pancreatic endodermlineage from pluripotent stem cells, comprising the steps of:

-   -   a. Culturing pluripotent stem cells,    -   b. Differentiating the pluripotent stem cells into cells        expressing markers characteristic of the definitive endoderm        lineage, and    -   c. Differentiating the cells expressing markers characteristic        of the definitive endoderm lineage into cells expressing markers        characteristic of the pancreatic endoderm lineage.

In one aspect of the present invention, the cells expressing markerscharacteristic of the pancreatic endoderm lineage co-express PDX1,NKX6.1, but do not express CDX-2 and NGN3.

Differentiation of Pluripotent Stem Cells into Cells Expressing MarkersCharacteristic of the Definitive Endoderm Lineage

Formation of cells expressing markers characteristic of the definitiveendoderm lineage may be determined by testing for the presence of themarkers before and after following a particular protocol. Pluripotentstem cells typically do not express such markers. Thus, differentiationof pluripotent cells is detected when cells begin to express them.

Pluripotent stem cells may be differentiated into cells expressingmarkers characteristic of the definitive endoderm lineage by any methodin the art or by any method proposed in this invention.

For example, pluripotent stem cells may be differentiated into cellsexpressing markers characteristic of the definitive endoderm lineageaccording to the methods disclosed in D'Amour et al, NatureBiotechnology 23, 1534-1541 (2005).

For example, pluripotent stem cells may be differentiated into cellsexpressing markers characteristic of the definitive endoderm lineageaccording to the methods disclosed in Shinozaki et al, Development 131,1651-1662 (2004).

For example, pluripotent stem cells may be differentiated into cellsexpressing markers characteristic of the definitive endoderm lineageaccording to the methods disclosed in McLean et al, Stem Cells 25, 29-38(2007).

For example, pluripotent stem cells may be differentiated into cellsexpressing markers characteristic of the definitive endoderm lineageaccording to the methods disclosed in D'Amour et al, NatureBiotechnology 24, 1392-1401 (2006).

For example, pluripotent stem cells may be differentiated into cellsexpressing markers characteristic of the definitive endoderm lineage byculturing the pluripotent stem cells in medium containing activin A inthe absence of serum, then culturing the cells with activin A and serum,and then culturing the cells with activin A and serum of a differentconcentration. An example of this method is disclosed in NatureBiotechnology 23, 1534-1541 (2005).

For example, pluripotent stem cells may be differentiated into cellsexpressing markers characteristic of the definitive endoderm lineage byculturing the pluripotent stem cells in medium containing activin A inthe absence of serum, then culturing the cells with activin A with serumof another concentration. An example of this method is disclosed inD'Amour et al, Nature Biotechnology, 2005.

For example, pluripotent stem cells may be differentiated into cellsexpressing markers characteristic of the definitive endoderm lineage byculturing the pluripotent stem cells in medium containing activin A anda Wnt ligand in the absence of serum, then removing the Wnt ligand andculturing the cells with activin A with serum. An example of this methodis disclosed in Nature Biotechnology 24, 1392-1401 (2006).

For example, pluripotent stem cells may be differentiated into cellsexpressing markers characteristic of the definitive endoderm lineage bytreating the pluripotent stem cells according to the methods disclosedin U.S. patent application Ser. No. 11/736,908, assigned to LifeScan,Inc.

For example, pluripotent stem cells may be differentiated into cellsexpressing markers characteristic of the definitive endoderm lineage bytreating the pluripotent stem cells according to the methods disclosedin U.S. patent application Ser. No. 11/779,311, assigned to LifeScan,Inc.

For example, pluripotent stem cells may be differentiated into cellsexpressing markers characteristic of the definitive endoderm lineage bytreating the pluripotent stem cells according to the methods disclosedin U.S. patent application Ser. No. 60/990,529.

For example, pluripotent stem cells may be differentiated into cellsexpressing markers characteristic of the definitive endoderm lineage bytreating the pluripotent stem cells according to the methods disclosedin U.S. patent application Ser. No. 61/076,889.

For example, pluripotent stem cells may be differentiated into cellsexpressing markers characteristic of the definitive endoderm lineage bytreating the pluripotent stem cells according to the methods disclosedin U.S. patent application Ser. No. 61/076,900.

For example, pluripotent stem cells may be differentiated into cellsexpressing markers characteristic of the definitive endoderm lineage bytreating the pluripotent stem cells according to the methods disclosedin U.S. patent application Ser. No. 61/076,908.

For example, pluripotent stem cells may be differentiated into cellsexpressing markers characteristic of the definitive endoderm lineage bytreating the pluripotent stem cells according to the methods disclosedin U.S. patent application Ser. No. 61/076,915.

Differentiation of Cells Expressing Markers Characteristic of theDefinitive Endoderm Lineage into Cells Expressing Markers Characteristicof the Pancreatic Endoderm Lineage

In one embodiment, cells expressing markers characteristic of thedefinitive endoderm lineage are differentiated into cells expressingmarkers characteristic of the pancreatic endoderm lineage thatco-express PDX1, NKX6.1, but do not express CDX2 and NGN3, by culturingthe cells expressing markers characteristic of the definitive endodermlineage in a first medium supplemented with FGF7, followed by culturingthe cells in a second medium supplemented with FGF7, a factor capable ofinhibiting BMP, activin A, retinoic acid, and a hedgehog signalingpathway inhibitor.

In one embodiment, FGF7 may be used at a concentration from about 50pg/ml to about 50 μg/ml. In one embodiment, FGF7 is used at aconcentration of 50 ng/ml.

In one embodiment, the factor capable of inhibiting BMP is noggin.Noggin may be used at a concentration from about 500 ng/ml to about 500μg/ml. In one embodiment, noggin is used at a concentration of 100ng/ml.

Activin A may be used at a concentration from about 2 ng/ml to 100ng/ml. In one embodiment, activin A is used at a concentration of 20ng/ml. In an alternate embodiment, activin A is used at a concentrationof 50 ng/ml.

Retinoic acid may be used at a concentration from about 1 nM to about 1mM. In one embodiment, retinoic acid is used at a concentration of 1 μM.

In one embodiment, the hedgehog signaling pathway inhibitor iscyclopamine-KAAD. Cyclopamine-KAAD may be used at a concentration fromabout 0.025 μM to about 2.5 μM. In one embodiment, cyclopamine-KAAD isused at a concentration of 0.25 μM.

The efficiency of differentiation may be determined by exposing atreated cell population to an agent (such as an antibody) thatspecifically recognizes a protein marker expressed by cells expressingmarkers characteristic of the definitive endoderm lineage.

Methods for assessing expression of protein and nucleic acid markers incultured or isolated cells are standard in the art. These includequantitative reverse transcriptase polymerase chain reaction (RT-PCR),Northern blots, in situ hybridization (see, e.g., Current Protocols inMolecular Biology (Ausubel et al., eds. 2001 supplement)), andimmunoassays such as immunohistochemical analysis of sectioned material,Western blotting, and for markers that are accessible in intact cells,flow cytometry analysis (FACS) (see, e.g., Harlow and Lane, UsingAntibodies: A Laboratory Manual, New York: Cold Spring Harbor LaboratoryPress (1998)).

Characteristics of pluripotent stem cells are well known to thoseskilled in the art, and additional characteristics of pluripotent stemcells continue to be identified. Pluripotent stem cell markers include,for example, the expression of one or more of the following: ABCG2,cripto, FOXD3, CONNEXIN43, CONNEXIN45, OCT4, SOX2, Nanog, hTERT, UTF1,ZFP42, SSEA-3, SSEA-4, Tra 1-60, Tra 1-81.

After treating pluripotent stem cells with the methods of the presentinvention, the differentiated cells may be purified by exposing atreated cell population to an agent (such as an antibody) thatspecifically recognizes a protein marker, such as CXCR4, expressed bycells expressing markers characteristic of the definitive endodermlineage.

Pluripotent stem cells suitable for use in the present inventioninclude, for example, the human embryonic stem cell line H9 (NIH code:WA09), the human embryonic stem cell line H1 (NIH code: WA01), the humanembryonic stem cell line H7 (NIH code: WA07), and the human embryonicstem cell line SA002 (Cellartis, Sweden). Also suitable for use in thepresent invention are cells that express at least one of the followingmarkers characteristic of pluripotent cells: ABCG2, cripto, CD9, FOXD3,CONNEXIN43, CONNEXIN45, OCT4, SOX2, Nanog, hTERT, UTF1, ZFP42, SSEA-3,SSEA-4, Tra 1-60, and Tra 1-81.

Markers characteristic of the definitive endoderm lineage are selectedfrom the group consisting of SOX17, GATA4, HNF3 beta, GSC, CERT, Nodal,FGF8, Brachyury, Mix-like homeobox protein, FGF4 CD48, eomesodermin(EOMES), DKK4, FGF17, GATA6, CXCR4, C-Kit, CD99, and OTX2. Suitable foruse in the present invention is a cell that expresses at least one ofthe markers characteristic of the definitive endoderm lineage. In oneaspect of the present invention, a cell expressing markerscharacteristic of the definitive endoderm lineage is a primitive streakprecursor cell. In an alternate aspect, a cell expressing markerscharacteristic of the definitive endoderm lineage is a mesendoderm cell.In an alternate aspect, a cell expressing markers characteristic of thedefinitive endoderm lineage is a definitive endoderm cell.

Markers characteristic of the pancreatic endoderm lineage are selectedfrom the group consisting of PDX1, HNF1 beta, PTF1 alpha, HNF6, HB9 andPROX1. Suitable for use in the present invention is a cell thatexpresses at least one of the markers characteristic of the pancreaticendoderm lineage. In one aspect of the present invention, a cellexpressing markers characteristic of the pancreatic endoderm lineage isa pancreatic endoderm cell.

Formation of Cells Expressing Markers Characteristic of the PancreaticEndocrine Lineage

In one embodiment, the cells expressing markers characteristic of thepancreatic endoderm lineage that co-express PDX1, NKX6.1, but do notexpress CDX2 and NGN3, produced by the methods of the present inventionmay be further differentiated into cells expressing markerscharacteristic of the pancreatic endocrine lineage.

Cells expressing markers characteristic of the pancreatic endodermlineage may be differentiated into cells expressing markerscharacteristic of the pancreatic endocrine lineage by any method in theart or by any method proposed in this invention.

For example, cells expressing markers characteristic of the pancreaticendoderm lineage obtained according to the methods of the presentinvention are further differentiated into cells expressing markerscharacteristic of the pancreatic endocrine lineage, by culturing thecells expressing markers characteristic of the pancreatic endodermlineage in medium containing exendin 4, then removing the mediumcontaining exendin 4 and subsequently culturing the cells in mediumcontaining exendin 1, IGF-1 and HGF. An example of this method isdisclosed in D'Amour et al, Nature Biotechnology, 2006.

For example, cells expressing markers characteristic of the pancreaticendoderm lineage obtained according to the methods of the presentinvention are further differentiated into cells expressing markerscharacteristic of the pancreatic endocrine lineage, by culturing thecells expressing markers characteristic of the pancreatic endodermlineage in medium containing DAPT (Sigma-Aldrich, MO) and exendin 4. Anexample of this method is disclosed in D'Amour et al, NatureBiotechnology, 2006.

For example, cells expressing markers characteristic of the pancreaticendoderm lineage obtained according to the methods of the presentinvention are further differentiated into cells expressing markerscharacteristic of the pancreatic endocrine lineage, by culturing thecells expressing markers characteristic of the pancreatic endodermlineage in medium containing exendin 4. An example of this method isdisclosed in D'Amour et al, Nature Biotechnology, 2006.

For example, cells expressing markers characteristic of the pancreaticendoderm lineage obtained according to the methods of the presentinvention are further differentiated into cells expressing markerscharacteristic of the pancreatic endocrine lineage, by treating thecells expressing markers characteristic of the pancreatic endodermlineage with a factor that inhibits the Notch signaling pathway,according to the methods disclosed in U.S. patent application Ser. No.11/736,908, assigned to LifeScan, Inc.

For example, cells expressing markers characteristic of the pancreaticendoderm lineage obtained according to the methods of the presentinvention are further differentiated into cells expressing markerscharacteristic of the pancreatic endocrine lineage, by treating thecells expressing markers characteristic of the pancreatic endodermlineage with a factor that inhibits the Notch signaling pathway,according to the methods disclosed in U.S. patent application Ser. No.11/779,311, assigned to LifeScan, Inc.

For example, cells expressing markers characteristic of the pancreaticendoderm lineage obtained according to the methods of the presentinvention are further differentiated into cells expressing markerscharacteristic of the pancreatic endocrine lineage, by treating thecells expressing markers characteristic of the pancreatic endodermlineage with a factor that inhibits the Notch signaling pathway,according to the methods disclosed in U.S. patent application Ser. No.60/953,178, assigned to LifeScan, Inc.

For example, cells expressing markers characteristic of the pancreaticendoderm lineage obtained according to the methods of the presentinvention are further differentiated into cells expressing markerscharacteristic of the pancreatic endocrine lineage, by treating thecells expressing markers characteristic of the pancreatic endodermlineage with a factor that inhibits the Notch signaling pathway,according to the methods disclosed in U.S. patent application Ser. No.60/990,529, assigned to LifeScan, Inc.

Markers characteristic of the pancreatic endocrine lineage are selectedfrom the group consisting of NGN3, NEUROD, ISL1, PDX1, NKX6.1, PAX4,NGN3, and PTF-1 alpha. In one embodiment, a pancreatic endocrine cell iscapable of expressing at least one of the following hormones: insulin,glucagon, somatostatin, and pancreatic polypeptide. Suitable for use inthe present invention is a cell that expresses at least one of themarkers characteristic of the pancreatic endocrine lineage. In oneaspect of the present invention, a cell expressing markerscharacteristic of the pancreatic endocrine lineage is a pancreaticendocrine cell. The pancreatic endocrine cell may be a pancreatichormone-expressing cell. Alternatively, the pancreatic endocrine cellmay be a pancreatic hormone-secreting cell.

In one aspect of the present invention, the pancreatic endocrine cell isa cell expressing markers characteristic of the β cell lineage. A cellexpressing markers characteristic of the β cell lineage expresses PDX1and at least one of the following transcription factors: NGN3, NKX2.2,NKX6.1, NEUROD, ISL1, HNF3 beta, MAFA, PAX4, and PAX6. In one aspect ofthe present invention, a cell expressing markers characteristic of the βcell lineage is a β cell.

Therapies

In one aspect, the present invention provides a method for treating apatient suffering from, or at risk of developing, Type 1 diabetes. Inone embodiment, the method involves culturing pluripotent stem cells,differentiating the pluripotent stem cells in vitro into a β-celllineage, and implanting the cells of a β-cell lineage into a patient. Inan alternate embodiment, the method involves culturing pluripotent stemcells, differentiating the pluripotent stem cells in vitro into cellsexpressing markers characteristic of the pancreatic endoderm lineagethat co-express PDX1, NKX6.1, but do not express CDX2 and NGN3, andimplanting the cells of the pancreatic endoderm lineage that co-expressPDX1, NKX6.1, but do not express CDX2 and NGN3 into a patient.

In yet another aspect, this invention provides a method for treating apatient suffering from, or at risk of developing, Type 2 diabetes. Inone embodiment, the method involves culturing pluripotent stem cells,differentiating the pluripotent stem cells in vitro into a β-celllineage, and implanting the cells of a β-cell lineage into a patient. Inan alternate embodiment, the method involves culturing pluripotent stemcells, differentiating the pluripotent stem cells in vitro into cellsexpressing markers characteristic of the pancreatic endoderm lineagethat co-express PDX1, NKX6.1, but do not express CDX2 and NGN3, andimplanting the cells of the pancreatic endoderm lineage that co-expressPDX1, NKX6.1, but do not express CDX2 and NGN3 into a patient.

If appropriate, the patient can be further treated with pharmaceuticalagents or bioactives that facilitate the survival and function of thetransplanted cells. These agents may include, for example, insulin,members of the TGF-β family, including TGF-β1, 2, and 3, bonemorphogenic proteins (BMP-2, -3, -4, -5, -6, -7, -11, -12, and -13),fibroblast growth factors-1 and -2, platelet-derived growth factor-AA,and -BB, platelet rich plasma, insulin growth factor (IGF-I, II) growthdifferentiation factor (GDF-5, -6, -7, -8, -10, -15), vascularendothelial cell-derived growth factor (VEGF), pleiotrophin, endothelin,among others. Other pharmaceutical compounds can include, for example,nicotinamide, glucagon like peptide-I (GLP-1) and II, GLP-1 and 2mimetibody, Exendin-4, retinoic acid, parathyroid hormone, MAPKinhibitors, such as, for example, compounds disclosed in U.S. PublishedApplication 2004/0209901 and U.S. Published Application 2004/0132729.

The pluripotent stem cells may be differentiated into aninsulin-producing cell prior to transplantation into a recipient. In aspecific embodiment, the pluripotent stem cells are fully differentiatedinto β-cells, prior to transplantation into a recipient. Alternatively,the pluripotent stem cells may be transplanted into a recipient in anundifferentiated or partially differentiated state. Furtherdifferentiation may take place in the recipient.

Definitive endoderm cells or, alternatively, pancreatic endoderm cells,or, alternatively, β cells, may be implanted as dispersed cells orformed into clusters that may be infused into the hepatic portal vein.Alternatively, cells may be provided in biocompatible degradablepolymeric supports, porous non-degradable devices or encapsulated toprotect from host immune response. Cells may be implanted into anappropriate site in a recipient. The implantation sites include, forexample, the liver, natural pancreas, renal subcapsular space, omentum,peritoneum, subserosal space, intestine, stomach, or a subcutaneouspocket.

To enhance further differentiation, survival or activity of theimplanted cells, additional factors, such as growth factors,antioxidants or anti-inflammatory agents, can be administered before,simultaneously with, or after the administration of the cells. Incertain embodiments, growth factors are utilized to differentiate theadministered cells in vivo. These factors can be secreted by endogenouscells and exposed to the administered cells in situ. Implanted cells canbe induced to differentiate by any combination of endogenous andexogenously administered growth factors known in the art.

The amount of cells used in implantation depends on a number of variousfactors including the patient's condition and response to the therapy,and can be determined by one skilled in the art.

In one aspect, this invention provides a method for treating a patientsuffering from, or at risk of developing diabetes. This method involvesculturing pluripotent stem cells, differentiating the cultured cells invitro into a β-cell lineage, and incorporating the cells into athree-dimensional support. The cells can be maintained in vitro on thissupport prior to implantation into the patient. Alternatively, thesupport containing the cells can be directly implanted in the patientwithout additional in vitro culturing. The support can optionally beincorporated with at least one pharmaceutical agent that facilitates thesurvival and function of the transplanted cells.

Support materials suitable for use for purposes of the present inventioninclude tissue templates, conduits, barriers, and reservoirs useful fortissue repair. In particular, synthetic and natural materials in theform of foams, sponges, gels, hydrogels, textiles, and nonwovenstructures, which have been used in vitro and in vivo to reconstruct orregenerate biological tissue, as well as to deliver chemotactic agentsfor inducing tissue growth, are suitable for use in practicing themethods of the present invention. See, for example, the materialsdisclosed in U.S. Pat. Nos. 5,770,417, 6,022,743, 5,567,612, 5,759,830,6,626,950, 6,534,084, 6,306,424, 6,365,149, 6,599,323, 6,656,488, U.S.Published Application 2004/0062753 A1, U.S. Pat. Nos. 4,557,264and6,333,029.

To form a support incorporated with a pharmaceutical agent, thepharmaceutical agent can be mixed with the polymer solution prior toforming the support. Alternatively, a pharmaceutical agent could becoated onto a fabricated support, preferably in the presence of apharmaceutical carrier. The pharmaceutical agent may be present as aliquid, a finely divided solid, or any other appropriate physical form.Alternatively, excipients may be added to the support to alter therelease rate of the pharmaceutical agent. In an alternate embodiment,the support is incorporated with at least one pharmaceutical compoundthat is an anti-inflammatory compound, such as, for example compoundsdisclosed in U.S. Pat. No. 6,509,369.

The support may be incorporated with at least one pharmaceuticalcompound that is an anti-apoptotic compound, such as, for example,compounds disclosed in U.S. Pat. No. 6,793,945.

The support may also be incorporated with at least one pharmaceuticalcompound that is an inhibitor of fibrosis, such as, for example,compounds disclosed in U.S. Pat. No. 6,331,298.

The support may also be incorporated with at least one pharmaceuticalcompound that is capable of enhancing angiogenesis, such as, forexample, compounds disclosed in U.S. Published Application 2004/0220393and U.S. Published Application 2004/0209901.

The support may also be incorporated with at least one pharmaceuticalcompound that is an immunosuppressive compound, such as, for example,compounds disclosed in U.S. Published Application 2004/0171623.

The support may also be incorporated with at least one pharmaceuticalcompound that is a growth factor, such as, for example, members of theTGF-β family, including TGF-β1, 2, and 3, bone morphogenic proteins(BMP-2, -3,-4, -5, -6, -7, -11, -12, and -13), fibroblast growthfactors-1 and -2, platelet-derived growth factor-AA, and -BB, plateletrich plasma, insulin growth factor (IGF-I, II) growth differentiationfactor (GDF-5, -6, -8, -10, -15), vascular endothelial cell-derivedgrowth factor (VEGF), pleiotrophin, endothelin, among others. Otherpharmaceutical compounds can include, for example, nicotinamide, hypoxiainducible factor 1-alpha, glucagon like peptide-I (GLP-1), GLP-1 andGLP-2 mimetibody, and II, Exendin-4, nodal, noggin, NGF, retinoic acid,parathyroid hormone, tenascin-C, tropoelastin, thrombin-derivedpeptides, cathelicidins, defensins, laminin, biological peptidescontaining cell- and heparin-binding domains of adhesive extracellularmatrix proteins such as fibronectin and vitronectin, MAPK inhibitors,such as, for example, compounds disclosed in U.S. Published Application2004/0209901 and U.S. Published Application 2004/0132729.

The incorporation of the cells of the present invention into a scaffoldcan be achieved by the simple depositing of cells onto the scaffold.Cells can enter into the scaffold by simple diffusion (J. Pediatr. Surg.23 (1 Pt 2): 3-9 (1988)). Several other approaches have been developedto enhance the efficiency of cell seeding. For example, spinner flaskshave been used in seeding of chondrocytes onto polyglycolic acidscaffolds (Biotechnol. Prog. 14(2): 193-202 (1998)). Another approachfor seeding cells is the use of centrifugation, which yields minimumstress to the seeded cells and enhances seeding efficiency. For example,Yang et al. developed a cell seeding method (J. Biomed. Mater. Res.55(3): 379-86 (2001)), referred to as Centrifugational CellImmobilization (CCI).

The present invention is further illustrated, but not limited by, thefollowing examples.

EXAMPLE 1 Differentiation of Human Pluripotent Stem Cells into CellsExpressing Markers Characteristic of the Pancreatic Endoderm Lineagethat co-express PDX1, NKX6.1, but do not express CDX2 and NGN3

This example demonstrates that activin A can be used in combination withNoggin and retinoic acid to facilitate the up-regulation of NKX6.1expression. Briefly, cells of the human embryonic stem cell line H1 werecultured on MATRIGEL™ (1:30 dilution) coated dishes and RPMI mediumsupplemented with 2% BSA, 100 ng/ml activin A, 20 ng/ml WNT-3a, 8 ng/mlof bFGF for one day, followed by treatment with RPMI media supplementedwith 2% BSA, 100 ng/ml activin A, 8 ng/ml of bFGF for an additional twodays (Stage 1), then

-   -   a. DMEM/F12+2% BSA+50 ng/ml FGF7+0.25 μM Cyclopamine-KAAD for        three days (Stage 2), then    -   b. DMEM-High glucose+1% B27+50 ng/ml FGF7+0.25 μM        Cyclopamine-KAAD+2 μM Retinoic acid (RA)+100 ng/ml of Noggin+20        ng/ml activin A or 50 ng/ml activin A for four days (Stage 3).

As a control, separate populations of cells were treated with DMEM Highglucose, supplemented with 1% B27, 50 ng/ml FGF7, 0.25 μMCyclopamine-KAAD, 2 μM retinoic acid (RA), and 100 ng/ml of Noggin.

Cultures were sampled in duplicate on stage 3 day 4, and analyzed forexpression of pancreatic markers using real-time PCR.

As shown in FIG. 1, treatment with activin A evoked an increase ofNKX6.1 expression at stage 3 day 4, which was greater than that observedfrom cells receiving no activin A. The increase in expression of NKX6.1,mediated by activin A, increased in proportion to the activin A dose. Adown-regulation of NGN3 expression was also observed in the cells atstage 3 day 4.

To determine whether the TGF-beta pathway was involved in facilitatingthe formation of cells expressing markers characteristic of thepancreatic endocrine lineage that co-express PDX1, NKX6.1, but do notexpress CDX2 and NGN3, cells were treated as follows: Cells of the humanembryonic stem cell line H1 were cultured on MATRIGEL™-coated plates(1:30 dilution), and differentiated into using the following protocol:

-   -   a. RPMI medium (Catalogue #22400, Invitrogen, Ca) supplemented        with 2% BSA (Catalog #152401, MP Biomedical, Ohio), and 100        ng/ml activin A (R&D Systems, MN) plus 20 ng/ml WNT-3a (Catalog        #1324-WN-002, R&D Systems, MN) plus 8 ng/ml of bFGF (Catalog        #100-18B, PeproTech, NJ), for one day followed by treatment with        RPMI media supplemented with 2% BSA and 100 ng/ml activin A plus        8 ng/ml of bFGF for an additional two days (Stage 1), then    -   b. DMEM/F12 (Catalogue #11330, Invitrogen, Ca)+2% BSA+50 ng/ml        FGF7 for three days (Stage 2), then    -   c. Either Treatment 1: DMEM (high glucose)+1% B27 (Invitrogen,        CA)+50 ng/ml FGF7, 0.25 μM Cyclopamine-KAAD, 2 μM retinoic acid        (RA) and 100 ng/ml of Noggin for four days, or    -   d. Treatment 2: DMEM (high glucose)+1% B27 (Invitrogen, CA)+50        ng/ml FGF7, 0.25 μM Cyclopamine-KAAD, 2 μM retinoic acid (RA),        100 ng/ml of Noggin, 20 ng/ml activin A, for four days, or    -   e. Treatment 3: DMEM (high glucose)+1% B27 (Invitrogen, CA)+50        ng/ml FGF7, 0.25 μM Cyclopamine-KAAD, 2 μM retinoic acid (RA),        100 ng/ml of Noggin, 1 μM ALK5 inhibitor II, for four days.

Cultures were sampled in duplicate on stage 3 day 4, and analyzed forexpression of pancreatic markers using real-time PCR. Cultures also werefixed in parallel for immunofluorescence analysis.

Table 1 shows the relative expression levels of NKX6.1, NGN3 and PDX1 atstage 3 day 4 when normalized to the most minimal condition in thisexperiment (treatment 1).

TABLE 1 NGN3 NKX6.1 PDX1 Treatment 1 1 1 1 Treatment 2 0.02 5.97 1.13Treatment 3 5.64 0.02 0.65

Treatment 1 (FGF7, retinoic acid and Noggin) induced the expression ofNKX6.1 and NGN3. See FIG. 2, panels a and b. However, the addition ofactivin A (treatment 2) blocked the expression of NGN3, andsignificantly increased the number of NKX6.1 expressing cells. See FIG.2, panels c and d. These data suggest that activation of the TGFβreceptor pathway during the formation of a population of cellsexpressing markers characteristic of the pancreatic endoderm lineageresults in a population of cells expressing markers characteristic ofthe pancreatic endoderm lineage that do not express NGN3.

Incubation of cells with the TGFβ receptor inhibitor Alk5 inhibitor IIconfirmed this hypothesis (see Treatment 3). Treatment of cells in DMEM(high glucose) supplemented with 1% B27 (Invitrogen, CA), 50 ng/ml FGF7,0.25 μM Cyclopamine-KAAD, 2 μM retinoic acid (RA), 100 ng/ml of Noggin,1 μM ALK5 inhibitor II resulted in a decrease in the level of expressionof NKX6.1. The level of expression observed was lower than that observedin cells that received treatment 1. See Table 1, and FIG. 2, panel e. Onthe other hand, the number of NGN3 expressing cells was significantlyincreased. See Table 1, and FIG. 2, panel f. No significant impact onthe PDX1 expression was observed. These results suggest that thecombination of Noggin, retinoid acid and activin A acts synergisticallyto specify a pancreatic precursor cell population that is positive forthe expression of NKN6.1 and PDX1, but negative for the expression ofNGN3.

As shown in FIG. 3, panels a and b, most PDX1 expressing cells generatedby using DMEM (Treatment 2—DMEM (high glucose)+1% B27 (Invitrogen,CA)+50 ng/ml FGF7, 0.25 μM Cyclopamine-KAAD, 2 μM retinoic acid (RA),100 ng/ml of Noggin, 20 ng/ml activin A) did not express CDX2 at stage 3day 4. This is in contrast with PDX1 expressing cells generated by usingDMEM/F12 supplemented with 1% B27 (Invitrogen, CA)+50 ng/ml FGF7, 0.25μM Cyclopamine-KAAD, 2 μM retinoic acid (RA), 100 ng/ml of Noggin, 20ng/ml activin A, wherein most PDX1 expressing cells also expressed CDX2.See FIG. 3, panels c and d.

Publications cited throughout this document are hereby incorporated byreference in their entirety. Although the various aspects of theinvention have been illustrated above by reference to examples andpreferred embodiments, it will be appreciated that the scope of theinvention is defined not by the foregoing description but by thefollowing claims properly construed under principles of patent law.

What is claimed is:
 1. A method to differentiate a population of humanpluripotent stem cells into a population of pancreatic endoderm cellsthat co-express PDX-1 and NKX-6.1, but do not express CDX-2 and NGN-3comprising the steps of: (a) culturing human pluripotent stem cells, (b)differentiating the human pluripotent stem cells into definitiveendoderm cells by treating the human pluripotent stem cells with amedium supplemented with a TGF-β receptor agonist, (c) differentiatingthe definitive endoderm cells into a population of pancreatic endodermcells that co-express PDX-1 and NKX6.1, but do not express CDX2 and NGN3by; i) culturing the definitive endoderm cells in a first mediumsupplemented with FGF7, followed by ii) culturing the cells from step i)in a second medium supplemented with FGF7, a factor capable ofinhibiting BMP, activin A, retinoic acid, and a hedgehog signalingpathway inhibitor, and (d) selecting cells which co-express PDX-1 andNKX6.1 and do not express CDX2 and NGN3 to obtain a purified populationof pancreatic endoderm cells that co-express PDX-1 and NKX-6.1, but donot express CDX-2 and NGN-3.
 2. The method of claim 1, wherein thefactor capable of inhibiting BMP is noggin.
 3. The method of claim 1,wherein the hedgehog signaling pathway inhibitor is cyclopamine-KAAD. 4.A method of differentiating definitive endoderm cells into a purifiedpopulation of pancreatic endoderm cells that co-express PDX-1 andNKX6.1, but do not express CDX2 and NGN3 by: (a) treating definitiveendoderm cells with a first medium supplemented with FGF7; and (b)culturing the treated definitive endoderm cells in a second mediumsupplemented with FGF7, a factor capable of inhibiting BMP, activin A,retinoic acid, and a hedgehog signaling pathway inhibitor to obtain apopulation of pancreatic endoderm cells that co-express PDX-1 andNKX-6.1, but do not express CDX-2 and NGN-3, and (c) selecting cellswhich co-express PDX-1 and NKX6.1 and do not express CDX2 and NGN3 toobtain a purified population of pancreatic endoderm cells thatco-express PDX-1 and NKX-6.1, but do not express CDX-2 and NGN-3.